WO2007028981A1

WO2007028981A1 - Apparatus and method for sonoporation

Info

Publication number: WO2007028981A1
Application number: PCT/GB2006/003291
Authority: WO
Inventors: Paul A. Campbell; Paul A. Prentice
Original assignee: University Of Dundee
Priority date: 2005-09-08
Filing date: 2006-09-07
Publication date: 2007-03-15

Abstract

An apparatus and method for making or perforating a surface in which a ultrasonically rupturable material such as a microbubble is tethered to the surface to be marked or perforated and an ultrasound signal applied in order to rupture the material. The tether is constructed to retain the rupturable material at a predetermined distance from the surface.

Description

Apparatus and Method for Sonoporation

The present invention relates to an apparatus and method for sonoporation and in particular to an apparatus and method that allows a surface such as a cell membrane to be marked or perforated by the application of ultrasound.

The cell membrane represents the outer extremity of all eukaryotic cells. In mammals, it is essentially constituted by a thin (5nm) bi-layer film of lipids which enclose the cell, defining its boundary and maintaining the essential physical and chemical differences between the internal cytoplasm and the extra-cellular environment. Under normal circumstances, the lipid nature of the cell membrane acts as an impermeable barrier to the passage of most water soluble molecules. Thus, the selective introduction of therapeutic agents to the inside of dysfunctional or diseased cells is challenging.

In addressing this challenge, biophysical approaches offer an attractive route for generic drug delivery in that they typically offer wider applicability when compared with their viral or biochemical counterparts, which tend to be cell/tissue selective and often have serious side-effects.

It is known that the level of contrast in an ultrasound image can be increased in the presence of microbubbles . Microbubbles are typically hollow capsules that can be filled with a suitable gas and which have a diameter of the order of a few microns. The injection of microbubbles into a patient is known to improve the contrast between different features contained in the image.

It is also known that ultrasound exposure (insonation) in the presence of contrast agent microbubbles will enhance membrane permeability and lead to molecular uptake from the locale. Under higher ultrasound pressures (>0.2MPa), this process (sonoporation) can elicit a number of clinically relevant biological effects. Two specific therapeutic applications of sonoporation have been identified. Firstly, sonoporation has been used to kill cells by either direct physical lysis (lethal sonoporation) or by initiation of programmed cell death and secondly to deliver therapeutic agents and plasmid DNA whilst retaining cell viability. In addition, promising observations of tumour regression have been demonstrated in murine studies. Sonoporation thus offers significant therapeutic potential across the spectrum of disorders.

The use of sonoporation has been limited by the inability to understand and therefore control the mechanism by which sonoporation occurs. It is an object of the present invention to provide improvements in and relating to sonoporation.

In accordance with a first aspect of the invention there is provided an apparatus for marking or perforating a surface, the apparatus comprising: a sonoporation means attached to a tether, the tether being attachable to the surface.

Preferably the tether is of a predetermined length to allow the sonoporation means to be positioned at a predetermined distance from the surface.

Preferably the sonoporation means is a microbubble.

Preferably the tether means is a ligand adapted to bind to the surface.

Preferably, the microbubble is filled with a gas.

Preferably, the microbubble is an ultrasound contrast agent.

Preferably, the microbubble has a diameter of less than 20 microns.

Preferably, the microbubble has a diameter of between 1 and 10 microns.

Preferably, the microbubble has an albumin shell. Preferably, the microbubble exhausts a microjet of gas which forces the bubble shell into the tissue plane to gently rupture the cell membrane in a reversible manner.

Preferably, at higher ultrasound pressure amplitudes, the ultrasound stimulated microbubble forms an energetic hydrodynamic fluid microjet that punctures the membrane irreversibly leading to cell death.

Preferably, the microbubble undergoes quasi-spherical expansion to mark or perforate the surface.

In accordance with a second aspect of the invention there is provided an apparatus for sonoporation, the apparatus comprising: an ultrasound source; and tethering means for attaching one or more sonoporation means to a surface; wherein the ultrasound source is adapted to produce an ultrasound signal capable of rupturing the one or more sonoporation means.

Preferably the sonoporation means is a microbubble.

Preferably, the tethering means is of a predetermined length to allow the sonoporation means to be positioned at a predetermined distance from a surface.

Preferably, the predetermined length is sized to allow rupture of the sonoporation means to kill the cell. Optionally, the predetermined length is sized to allow rupture of the sonoporation means to produce a repairable hole in the cell.

Preferably, the tethering means is a ligand adapted to bind to a ligand binding site on the surface.

Preferably the ligand binding site is a cell adhesion molecule.

Preferably, the microbubbles are filled with a gas.

Preferably, the microbubbles are ultrasound contrast agents.

Preferably, the microbubbles have a diameter of less than 20 microns.

Preferably, the microbubbles have a diameter of between 1 and 10 microns.

Preferably, the microbubbles have an albumin shell.

Preferably, the microbubbles emit a microjet of fluid which etch, puncture or mark the surface.

Preferably, the microbubbles undergo quasi-spherical expansion to etch, puncture or mark the surface.

Preferably, the ruptured shells of the microbubbles etch, puncture or mark the surface. Preferably, the ultrasound source is capable of emitting ultrasound at a frequency of 0.9 to 1.1 MHz.

Preferably, the ultrasound source is capable of emitting ultrasound at a peak negative pressure of 0.1 to 10 MPa.

More preferably, the ultrasound source is capable of emitting ultrasound at a peak negative pressure of 0.5 to 5MPa.

The sonoporation means may be coated with a lipid mono- layer to assist with attaching the tether to the microbubble .

Optionally, covalent attachment of the tether to the sonoporation means is provided by incorporating a conjugate molecule in the microbubble shell.

Optionally, the conjugate molecule may be a lipid dermative molecule.

Optionally, the conjugate may be a carboxyl molecule.

Most preferably, the ultrasound source is capable of emitting ultrasound at a peak negative pressure of 0.2 to 1.6MPa .

Preferably, the depth of sonoporation is determined by the magnitude of the peak negative pressure.

Preferably, the ultrasound source is capable of emitting ultrasound in pulses. Preferably, the ultrasound pulses have a period of less than 250ms.

Preferably, the ultrasound source is coupled to the microbubbles by means of a liquid.

Preferably, the liquid is water.

The concentration of the microbubbles is set to minimise the extent of Bjerknes coupling of the microbubbles.

In accordance with a third aspect of the invention there is provided a method for etching, puncturing or marking a surface, the method comprising the steps of: attaching one or more sonoporation means at a predetermined distance from the surface by means of a tether; coupling an ultrasound source to said one or more sonoporation means; and rupturing the one or more sonoporation means using the ultrasound signal, the rupturing of the one or more sonoporation means causing the surface to be etched, punctured or marked.

Preferably each of the tethers comprises a ligand.

Preferably the step of positioning one or more sonoporation means comprises the step of attaching the ligand of at least one of the sonoporation means to at least one ligand binding site on the surface.

Preferably the sonoporation means is a microbubble. Preferably the surface is a cell membrane.

Preferably the ligand binding site comprises a cell adhesion molecule.

Preferably, the sonoporation means emits a microjet of gas which etches, punctures, or marks the surface.

Preferably, the sonoporation means undergoes quasi- spherical expansion to etch, puncture or mark the surface.

Preferably the sonoporation means is a microbubble.

Preferably, the ruptured shells of the microbubbles etch, puncture or mark the surface.

Preferably, the ultrasound signal is emitted at a frequency of 0.9 to 1.1 MHz.

Preferably, the ultrasound signal is emitted at a peak negative pressure of 0.1 to 10 MPa.

More preferably, the ultrasound signal is emitted at peak negative pressure of 0.5 to 5MPa

Most preferably, the ultrasound signal is emitted at peak negative pressure of 0.2 to 1.6MPa.

Preferably, the depth of etch, mark or perforation is determined by the magnitude of the peak negative pressure. Preferably, the ultrasound signal is emitted in pulses.

Preferably, the ultrasound pulses have a period of less than 100 s.

Preferably, the ultrasound source is coupled to the πiicrobubbles by means of a liquid.

Preferably, the liquid is transparent.

Preferably the liquid is water.

The present invention will now be described by way of example only with reference to the accompanying drawings in which:

Fig. 1 is a schematic drawing of a microbubble positioned at a predetermined distance from a cell by means of a ligand;

Fig. 2 (a) to 2 (d) illustrate schematically the sonoporation of a cell membrane determined by the ligand length;

Fig.3 is a series of frames showing a trapped microbubble;

Fig.4 is a series of frames showing microbubble cavitation;

Fig.5 is a frame showing microbubble cavitation; Fig.β is a frame of a pit formed in a surface caused by a microjet at high pressure;

Fig.7 is a frame of a pit formed in a surface caused by a microjet at low pressure;

Fig.8 is a series of frames showing a pit formed in a surface caused by a microjet at low pressure;

Fig.9 is a series of frames showing microbubble cavitation in proximity to a cell monolayer; and

Fig.10a to 1Of show the correlation of specific cavitation events with membrane damage.

Figure 1 shows an apparatus 1 in accordance with the present invention comprising a microbubble 3 that has been positioned at a predetermined distance from a cell 5 by means of an appropriately selected ligand 7. The predetermined distance is chosen to maximise a particular effect that rupturing the microbubble 3 will have on the cell 5. This effect might be perforation of the cell membrane, or indeed complete destruction of the cell.

The cell itself (as shown) comprises a cell membrane, which separates the intracellular space from the extracellular space, and governs what is able to move in and out of the cell. Located across the cell membrane is a cell adhesion protein 9, which extends from the intracellular space 21 to the extracellular space as is typical for such proteins. The extracellular portion of the cell adhesion protein 9 is capable of bonding to other molecules. The microbubble 3 consists of a denatured albumin shell containing a gas core. The gas, for example, may be octafluoropropane, and the diameter of the microbubble is typically in the range of 2 to 7 microns. However, the microbubble may be sized dependent on the desired effect of rupturing the microbubble on the targeted cell .

The microbubble 3 is connected to an appropriately selected ligand 7, the ligand 7 being a small molecule capable of binding to a larger macromolecule, in this case the ligand 7 is chosen to bind to the particular cell adhesion protein 9 of the cell 5. In another example the ligand 7 and the cell adhesion protein 9 may be intermediately linked by catenin proteins.

Rupturing a microbubble results in a jetting of the contents in what has been found to be a directional jet. This jet may impact on the cell and rupture the cell membrane. A sonopore is a hole in the cell membrane which is created by such a process, and the contents of the jet may thus enter the cell. Such an injection process leaves a sonopore which may remain open for several seconds. Large sonopores may be created which can be repaired provided the cell nucleus is not damaged. This provides a method of targeted injection of, for example, a molecule, into the cell.

Figure 2 illustrates this schematically. Fig.2 (a) shows a microbubble 3,- held at a predetermined distance D 11 from a cell membrane 5. A coupling medium 10 surrounds the cell and the attached microbubble. An ultrasonic wave 8, generated by a transmitter 6, travels through the coupling medium 10, preferably water, and impinges on the microbubble, illustrated in Fig.2(b). The ultrasonic wave 8 is selected so as to be resonant with the resonance frequency of the microbubble 3.

Under the influence of the ultrasonic wave 8, the microbubble 3 explosively ruptures 15, as shown in Fig.2(c), jetting the contents 13 in a directional manner towards the cell membrane 5. The force exerted on the cell membrane 5 by the jetted contents 13 results in rupture of the membrane 5. Generally, this results in creation of a hole 17, as illustrated in Fig.2(d). This hole 17 allows for flow of material from the extracavity space 19 into the intracavity space 21, and vice versa. This means that, for example, a drug may be injected into the cell without the need for a microneedle or other such apparatus .

The size of the hole is determined by the size of the microbubble, by the length of the ligand, which determines the distance between the microbubble and the cell membrane and the power of the ultrasound wave. Appropriate ligands can be chosen depending on the separation deemed appropriate for a particular sonoporation event. For example, a deep, but highly localised hole may be created by using a short ligand. This is likely to kill a cell.

Alternatively, a less intrusive and thus reparable hole can be made by using a longer tether for the same ultrasonic pressure conditions. In the meantime the remnants of the microbubble, namely the shell, are propelled ballistically towards the cell membrane in accordance with the laws of conservation of momentum. The shell impinges on the cell, stretching it beyond the rupture threshold causing it to become permeabilised.

It is also proposed that many microbubbles may be attached to a targeted cell-group. Such a cell-group that may be targeted could be a tumour or other disease related cells in the body of a patient, where the purpose would be to destroy the cell.

The major advantage of the invention is that the tethering of microbubbles to cells using appropriate ligands allows for controlled sonoporation. Rupturing the microbubbles can temporarily puncture, deliver controlled amounts of a molecule, or even destroy the cells. Using a known ligand allows the' distance between microbubble and cell membrane to be known relatively accurately and thus the effects of rupturing the microbubbles can be accurately predicted.

Preferably the ligand bound tether is stiff enough that buoyancy forces are overcome and orientation within a gravity field plays no role in the microbubble stand-off displacement.

In Figs. 3 to 7, representative ultra high speed sequences acquired at a framing rate of 50OkHz showing microbubble cavitation in proximity to naked coverslips (frames 43, 45, 47, 49, 51, 53, 55 and 57). Timings (in microseconds) relative to the instant of cavitation inception are indicated at each frame. Images were spatially calibrated by observing 6μm calibration beads (12 such beads have been superimposed on the scene to provide scale in frame 47) . Each frame measures 163 μm x 110 μm. Initially (t<0) a 4.5μm diameter microbubble 44 is trapped and manipulated to a displacement (D) 40, 2β.5μm from the coverslip (darker region to the right) . The cavitating bubble expands and microjet formation initiates with subsequent collapse.

These figures illustrate the ability to control sonoporation at a predetermined distance from a surface. The apparatus and method of the present invention uses a tether to position a microbubble at said predetermined distance from the surface, the tether being attachable to the surface.

In the sequence of Fig. 4, by controlled displacement of a similarly sized microbubble 54 to a location some 19μm farther from the substrate than shown in Fig.3, then insonating, only the microjet is seen to touch-down at the surface (at 6μs) .

A 10 x lOμm image of a sono-lithography pit 59 formed by microjet touchdown at high pressure is shown in Fig. 6. At lower pressures, much smaller features can be controllably written to the substrate. Fig.6 shows a lOOnm width pit 61 formed with a depth of just 25nm. Fig.8 shows this feature 63 in cross-section 65.

As is apparent in Figs. 3 to 7, upon cavitation inception, the microbubble 44 expands rapidly to give the expanded microbubble 46 with spontaneous formation of a thin (micrometer width) linear involuted microjet traversing its breadth and directed orthogonally towards the plane of the coverslip. The maximum distance in the z-axis direction at which the microjet will impinge upon the surface is defined by R_MMC. Accordingly, control of the z-axis direction displacement of the microbubble is used to refine the microbubble' s interaction with the substrate, so that microjet touchdown without contact of the expanded UCA shell can occur (Fig. 4) . This fine z- control could be used over a range of substrate moduli including cells, lithographic plates or the like. It is the control of the proximity of the microbubble with respect to the surface that allows the microjets to be preferentially directed towards the surface.

In the examples shown in Figs. 3 and 4, microjetting was relatively common on naked coverslips, occurring in 39% of this data. In the remaining cases the microbubbles engaged in quasi-spherical expansion and coverslip contact, without the formation of a clearly visible microjet.

Figs. 8 and 9 show ultra high speed sequences (frames 71, 73, 75, 79 and 81 (Fig.8) and 91, 93, 95, and 97 (Fig.9)) acquired at a framing rate of 50OkHz showing microbubble cavitation in proximity to cell monolayers.

In Figs. 8 and 9 the coverslips supported cultured monolayers of adherent live MCF7 (human breast cancer) cells at 70-80% confluence, in the sonoporation chamber containing Dulbeccos modified Eagle's medium (DMEM) . The dominant finding here was quasi-spherical expansion with contact, and compression of underlying cells 48%. In Fig.9, core ejection of gas was also observed during cavitation near cells. In this case, a βμm diameter microbubble 99 was initially trapped at a z-axis displacement of 19.5μm from a live cell and undergoes core ejection with resultant compression (frame 73) of the proximal cell as the shell remnant 107 recoils into it.

At later times the cell appears to partially recover its original morphology whereas the shell remnant 107 rebounds away from the cell and the ejected gas eventually reforms into a free bubble 105.

Core-ejection was observed in 35% of data acquired on monolayers. Here, the microbubble shell 99 appeared to rupture during the initial phase of cavitation, rapidly (within 2μs) giving rise to an emergent jet directed away from the coverslip plane. To conserve momentum, the shell remnant 107 is ballistically propelled towards the monolayer, compressing any intervening cells.

Assuming the entire membrane is stretched during this deformation process, and estimating the membrane areal increase ΔA, and its initial area A₀, the resultant areal strain can be calculated as (ΔA/A₀) . Approximating Ao « 2πR²(l-cos θ) , where R is the radius of curvature of the cell, and θ is the angle the cell membrane makes with the coverslip (θ «β2^s from Fig. 10, initial frame), then ΔA introduced by the impacting microbubble shell is given by πR_s ², where R_s=10μm (estimated from indentation depth in Fig. 10 at 4μs) is the radius of the hemispherical compression zone, recognised as the topological deformation of a circular patch of membrane also of radius R₃. ΔA/A₀ is thus calculated at 11.7%, exceeding the critical areal strain of between 2-5% previously reported to cause membrane rupture. A cell may thus be rendered temporarily permeable, by this process.

Active microjetting into monolayers was observed in 17% of such sequences. Here, microbubbles are seen to experience inertial cavitation and collapse, with the outermost hemisphere undergoing involution to form a central reentrant jet directed at the cell (Fig. 10b) .

With inter-frame times on the order of 0.5-1.0μs, a lower-bound estimate for the jet velocities, Vj , can be made, equating to circa 5.5ms^"1 (distance traversed (22μm) over traversal time (4μs) ) . Membrane breaching may occur if any induced tension exceeds the critical rupture stress, τ_cri_t, related to the local elastic modulus, E, thus:

tcrit = E . ε_r (D -

Here ε_r is the relative deformation (critical areal strain) needed for rupture to occur, a value commonly accepted²³ as 3%, hence ε_r=0.03. Measurements of E on live cells are consistently below lOOkPa leading to an approximate upper limit of τ_cri_t <* 3kPa. Consideration of two distinct candidate mechanisms: development of a water hammer pressure, P_m; and momentum exchange due to fluid flow, P_f, allows estimates of their respective induced pressures to be ascertained.

As P_NH * 0.5 p c Vj , where p is the fluid density (998 kg irf³) and c is the speed of sound in the medium (1480ms^"1) we thus estimate P_m * 4MPa. We estimated the flow induced pressure at the stagnation point as 0.5 p Vj², and therefore P_f » 15kPa, so that both pressure estimates exceed t_Cri_t/ confirming that either (or both) are viable breaching mechanisms in this high MI regime .

In Fig.10, correlation of specific cavitation events with membrane damage was observed using ultra high speed imaging and atomic force microscopy (AFM). Fig.11a is a 163μm x HOμm frame showing a quiescent 4μm diameter microbubble 114 trapped 17μra from a cell membrane 116. The shadow to the lower right of a (labelled 'N') is a notch 115 on the edge of the coverslip that facilitates rapid registration of the region of interest for post- insonation microscopy. Fig.10b shows that at t=8μs after cavitation inception, the microbubble has developed an involution to form a central jet which contacts the membrane 116 over a region some 15μm wide. By subsequent extraction of the insonated coverslip (within circa 20 seconds) , and chemically fixing in 4% paraformaldehyde (on ice) , the instantaneous membrane topography was preserved.

Fig.10c shows an atomic force micrograph frame 121 of fixed cells. In this case, the local topography near to the notched coverslip edge 115 (labelled *N^; ) aids identification of the cell that had been contacted by the microjet. The resultant sonopore 123 measures 16μm, as indicated on the respective cross-sections of Fig. 1Oe. A perspective view is shown of another sonopore 125, the cross-section through which is shown in Fig.1Of .

Pits (sonopores) were consistently observed on all cells previously located directly below the hypocentre of trapped microbubble that underwent microjetting into the respective cell. Fig. 10c shows that sonopore 123 formed due to the jetting event depicted in Figs. 10a-b. The cross-section through this entity is similar to that observed during UHS imaging of the contact phase of the microjet (Fig. 10b) .

Moreover, the jet's penetration power was such that sonopore depth extends to the underlying coverslip. Figure 1Od shows a perspective AFM image of a representative sonopore 125 formed on a different cell monolayer and exhibiting a distinct peripheral lip of raised material .

The spatial extent of these microjet induced sonopores suggests that such events probably lead to cell lysis. However, if damage to the nucleus, and vital organelles is avoided, then even large membrane disruptions, up to lOOOμm², can reseal, and a cell might remain viable after microjet penetration.

In this latter circumstance, it is pertinent to consider the level of molecular uptake possible during microjet injection. The volume of ambient fluid within a jet, Vj , can be approximated as Vj ~ O.1R_C ³, where R_c is the radius of a microbubble that is on the verge of collapsing. In our observations (e.g. Fig. 9), R_c * 27μm, leading to a microinjection volume of 2.0 picolitres .

In the case of the present invention micro-injection into the cells occurs and the membrane breach caused by the micro-injection can remain open for several seconds, allowing cytosol borne material to leak out of the cell. The use of optical trapping for initial positioning of the cells in a holographically generated array together with their microbubble counterparts which can be held in a separate LG trap array is further described below. The Gaussian traps are filled with cells within an optically gated reservoir (adjoining a main fluidics channel) , then the LG traps are filled with microbubbles from a separate optically gated reservoir.

The present invention provides an apparatus and method by which the energetic micrometer scale interactions between individual cells and cavitating microbubbles can be controlled by controlling the z-axis displacement of the microbubbles from the surface.

Of the handful of feasible biophysical approaches devised thus far, the use of ultrasound to mediate molecular delivery (i.e. sonoporation) has particular promise for clinical implementation as it offers the following possibilities:

(i) the treatment may not require open surgical access to the target tissues and thus patients would not be exposed to the trauma associated with incision nor the enhanced probability for subsequent infection; (ii) therapy may be implemented using standard hospital equipment with minor modifications; (iii) exposure is localised so that a target region only is affected.

Improvements and modifications may be incorporated herein without deviating from the scope of the invention herein intended. For example, any number of microbubbles may be tethered to a cell. Furthermore, the invention is not limited to cells, but may be applied to any surface to which a ligand may form a binding, be it for the purposes of sonoporation or for the purposes of partial or complete destruction.

Claims

1. An apparatus for marking or perforating a surface, the apparatus comprising: a sonoporation means attached to a tether, the tether being attachable to the surface.

2. An apparatus as claimed in claim 1 wherein, the tether is of a predetermined length to allow the sonoporation means to be positioned at a predetermined distance from the surface.

3. An apparatus as claimed in claim 1 or claim 2 wherein, the sonoporation means is a microbubble.

4. An apparatus as claimed in any preceding claim wherein, the tether is a ligand adapted to bind to the surface.

5. An apparatus as claimed in claim 3 wherein, the microbubble is filled with a gas.

6. An apparatus as claimed in claim 3 or claim 5 wherein, the microbubble is an ultrasound contrast agent.

7. An apparatus as claimed in claims 3, 5 or 6 wherein, the microbubble has a diameter of less than 20 microns.

8. An apparatus as claimed in claims 3 or 5 to 7 wherein, the microbubble has a diameter of between 1 and 10 microns.

9. An apparatus as claimed in claims 3 or 5 to 8 wherein, the microbubble has an albumin shell.

10. An apparatus as claimed in claims 3 or 5 to 9 wherein, the microbubble exhausts a microjet of gas which forces the bubble shell into the tissue plane to gently rupture the cell membrane in a reversible manner .

11. An apparatus as claimed in claims 3 or 5 to 10 wherein, at higher ultrasound pressure amplitudes, the ultrasound stimulated microbubble forms an energetic hydrodynamic fluid microjet that punctures the membrane irreversibly leading to cell death.

12. An apparatus as claimed in claims 3 or 5 to 11 wherein, the microbubble undergoes quasi-spherical expansion to mark or perforate the surface.

13. An apparatus as claimed in any preceding claims further comprising an ultrasound source.

14. An apparatus as claimed in claim 13 wherein, the ultrasound source is capable of emitting ultrasound at a frequency of 0.9 to 1.1 MHz.

15. An apparatus as claimed in claim 13 or 14 wherein, the ultrasound source is capable of emitting ultrasound at a peak negative pressure of 0.1 to 10 MPa.

16. An apparatus as claimed in claim 13 to 15 wherein, the ultrasound source is capable of emitting ultrasound at a peak negative pressure of 0.5 to 5MPa .

17. An apparatus as claimed in any preceding claim wherein, the sonoporation means is coated with a lipid mono-layer to assist with attaching the tether to the microbubble .

18. An apparatus as claimed in claim 17 wherein, covalent attachment of the tether to the sonoporation means microbubble is provided by incorporating a conjugate molecule in the microbubble shell.

19. An apparatus as claimed in claim 18 wherein, the conjugate molecule is a lipid dermative molecule.

20. An apparatus as claimed in claim 18 wherein, the conjugate is a carboxyl molecule.

21. An apparatus as claimed in any preceding claims wherein, the tether is a ligand adapted to bind to a ligand binding site on the surface.

22. An apparatus as claimed in claim 21 wherein, the ligand binding site is a cell adhesion molecule.

23. An apparatus as claimed in claims 13 to 16 wherein, the ultrasound source is capable of emitting ultrasound in pulses.

24. An apparatus as claimed in claims 13 to 16 or 23 wherein, the ultrasound pulses have a period of less than 250ms.

25. A method for etching, puncturing or marking a surface, the method comprising the steps of: attaching one or more sonoporation means at a predetermined distance from the surface by means of a tether; coupling an ultrasound source to said one or more sonoporation means; and rupturing the one or more sonoporation means using the ultrasound signal, the rupturing of the one or more sonoporation means causing the surface to be etched, punctured or marked.

26. The method as claimed in claim 25 wherein, the tether comprises a ligand.

27. The method as claimed in claims 25 and 26 wherein, the step of positioning one or more sonoporation means comprises the step of attaching the tether of at least one of the sonoporation means to at least one ligand binding sites on the surface.

28. An apparatus as claimed in claims 25 and 26 wherein, the sonoporation means emits a microjet of gas which etches, punctures, or marks the surface.

29. An apparatus as claimed in claims 25 to 27 wherein, the sonoporation means undergoes quasi-spherical expansion to etch, puncture or mark the surface.

30. An apparatus as claimed in claims 25 to 29 wherein, the ultrasound signal is emitted at a frequency of 0.9 to 1.1 MHz.

31. An apparatus as claimed in claims 25 to 30 wherein, the ultrasound signal is emitted at a peak negative pressure of 0.1 to 10 MPa.

32. An apparatus as claimed in claims 25 to 31 wherein, the ultrasound signal is emitted in pulses.